Gene expression using Gram-negative bacteria Allan R. Shatzman SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA Current Opinion in Biotechnology 1990, 1:5-11

Introduction The technology for expressing heterologous gene products has developed to the point where it may be successfully applied to almost any Gram-negative bacterial system. Yet, when one thinks of gene expression in Gramnegative bacteria, Escherichia coli is the organism that typically first comes to mind. E. coli has been, and continues to be, the workhorse of the gene expression field. No other system has been developed that can not only express a large number of known gene products at levels sufficient for detailed biochemical analysis or product development, but also express undefined coding sequences (open reading frames) in amounts sufficient to determine the identity of the gene product and to characterize its function. Indeed, literally hundreds of papers are published each year in which it has been reported that genes of prokaryotic or eukaryotic origin have been cloned and expressed in E. coil The ability to express this myriad of genes has been aided by the development of numerous expression vector systems which use highly efficient transcriptional and translational regulatory signals to optimize production to levels that may reach 30% of total cellular protein (for a review of expression vector technology see [ 1] ). However, over the last year or two, the focus of improving expression in E, coli has shifted from the optimization of these regulatory signals to the manipulation of the coding sequence itself and the quality of the protein being produced. This review highlights advances made over the last two years in our ability to optimize the expression of heterologous gene products in E. coli with respect to both the quantity and quality (solubility, folding, activity, and homogeneity) of the proteins being produced. In addition, it will spotlight our emerging abilities to express heterologous gene products in other Gram-negative organisms where the aim is not so much to overproduce these proteins but rather to impart a useful new phenotype or function to these organisms.

Optimization of gene expression via alterations in the coding sequence Initial efforts to express a heterologous gene in E. coli can often lead to less than desirable results with respect

to the quantity of protein produced. In some of these cases, even the use of several different transcriptional or translational regulatory signals does not improve the level of protein production. Several papers have now been published that demonstrate that production of such proteins can be dramatically improved by making alterations within the coding sequence of the gene under study. Seow et al. [2 " ] reported that expression of human tumor necrosis factor (TNF)-]3 could be increased from undetectable levels to 34% of total cell protein (TCP) by introducing silent mutations throughout the gene, which result in optimal codon utilization for E. coli, and also mutations in the 5' end of the gene, which minimize the development of secondary structure in that region of the message. Interestingly, expression levels were quite low (1-2% of TCP) if only the changes minimizing secondary structure were made, while no expression was detected when only the codon optimization changes were present. These results suggest that multiple mechanisms may be involved in obtaining alterations in expression levels, and that minimizing secondary structure in the translation initiation region may be somewhat more important than optimizing codon utilization. These findings are supported by Schauder and McCarthy [3 "] who have shown an inverse correlation between the expression of several genes and the degree of secondary structure present in the translation initiation region (e.g. expression is highest when the chance of secondary Structure in the 5' end of the message is lowest). In contrast, others have shown that a more random approach to coding sequence alterations can result in vastly improved expression levels. Devlin et al. [4 o.] increased expression of human granulocyte stimulating factor from undetectable levels to almost 20% of total soluble protein by altering all G and C residues in the 5' end of the gene to A and T without affecting the protein sequence. These changes actually greatly decreased the presence of preferred codons in this region of the gene and did not create any significant changes in mRNA secondary structure. Reduction in the GC content of the 5' end of the bovine growth hormone gene has also yielded increased expression levels in E. coli [5]. To further demonstrate the importance of alterations in the 5' end of a coding sequence (and to confuse the subject even more) we must turn our attention to the approach used by Isacchi et aL [6.-] to optimize expression of human apolipoprotein

Abbreviations BRP--bacteriocin release protein; M-CSF~macrophage colony-stimulating factor; RuBisCo~ribulose-l,5-bisphosphate carboxylase/oxygenase;TCP--total cell protein; TNF--tumor necrosis factor.

(~) Current Biology Ltd ISSN 0958-1669

5

6 Expressionsystems A1. After screening all the possible alternative sequences that did not change the protein sequence encoded by the 5' end of this gene, a clone was selected with expression levels that were greater than 100 times those obtained with the unaltered gene. Analysis of this clone showed no causal relationship between codon utilization, base composition, or alterations in secondary structure and expression levels. Similarly, dramatic increases have been observed in czl-antitrypsin expression after introduction of mutations that did not apparently improve codon utilization, reduce secondary structure, or significantly alter base composition [7]. In summary, while the mechanisms by which silent alterations in the 5' coding sequence affect expression are far from clear or consistent, the studies mentioned above prove that this is an important new parameter to be considered in optimizing gene expression.

Improved production of soluble and active proteins in E. coli Typically, the first concern in heterologous gene expression is how much can be made. However, after achiev ing a 'fat band on a gel' (which may correspond to a gram or more of product per liter), attention then turns to the utility of this massive amount of protein. There is indeed reason for serious concern as, unfortunately, most heterologous gene products expressed in E. coli are not produced in an active, properly folded, or soluble form. Instead, most recombinant proteins accumulate as insoluble aggregates or inclusion bodies in the cytoplasm of the bacteria. In some cases, this may actually be beneficial as these inclusion bodies are amenable to rapid purification and the protein may then be resolubilized and renatured to a fully active form. However, more often these insoluble products are ditticuk to recover in any quantity as fully active and soluble products. Considerable efforts have recently been made to understand what causes these proteins to be insoluble and to find ways of expressing these proteins in more soluble, active forms. Schein [8 o'] compiled a list of nine suggested reasons for the formation of inclusion bodies in E. coli which includes: excessively high rates of production, which do not allow sufficient time to achieve correct folding; high local concentrations of product leading to precipitation; and lack of post-translational modifying enzymes or foldases necessary to achieve the correct conformation. Recent advances in the production of soluble, active proteins using these considerations along with improvements in renaturing inactive products will be described below.

Optimization of product solubility by alterations in culture temperature Expression of subtilisin in E. coli using a high-expression vector under standard growth and induction con-

ditions was recently shown to lead to production of an insoluble product from which very little properly folded and active subtilisin could be obtained [9 "°]. However, when cells were grown at 23°C instead of 37°C, an increase in active subtilisin of up to 14-fold was achieved. Further studies showed that a reduction in the concentration of inducer used in this expression system could increase levels of active product even with growth at 37°C. When both temperature and inducer concentrations were adjusted, active product accumulated at levels 16-fold above those found under standard production conditions. These studies suggest that the rate at which a protein is made can affect its ability to fold properly. Similar effects of culture temperature on product solubility have been observed for the production of human interferon a2 [10°]. In these studies, overproduction of interferon at 37°C yielded only 5% soluble, active protein while growth at 28-30°C produced 73% soluble product. Thus, although total interferon production at 37°C was three times greater than at 28-30°C, the lower production temperature actually yielded a seven-fold increase in active product. To determine whether these differences in solubility were a result of changes in temperature or in concentration, the interferon gene was inserted into expression vectors that were known to express at lower levels of production. Regardless of absolute amount of product produced, expression at the lower temperature always yielded a higher percentage of soluble product and a greater yield of active interferon-~2. Similar increases in active product yield have also been reported for interferon-13 [11]. Seow et aL [2 "°] reported that expression at lower temperatures may have an additional benefit for optimizing product accumulation in E. coil In their studies with human TNF-I3, absolute levels of expression, as well as amounts of active protein, were several times higher at 25°C than at 37°C, despite the fact that the promoter used in their expression system (trp) is far more efficient at 37°C than at 25°C [12]. The authors suggest that product accumulation may be favored under conditions of reduced culture temperature because the level of proteolysis is reduced. Whether this is the case or not, these results all suggest that the best way to evaluate optimal expression in E. coli is by calculating the yield of fully active product, rather than by determining the more traditional value of grams of total product per liter.

Improved expression of soluble, active proteins via foldases and chaperonins Studies have been initiated recently to determine whether the co-expression of foldases along with the gene product of interest can result in improved solubility and activity. This approach is based on the observations that there are numerous proteins such as thioredoxin, protein disulfide isomerase, and prolyl cis-trans isomerase that seem to be designed to aid in protein folding. Although the usefulness of these proteins in improving product solubility in E. coli in vivo has not been documented

Gene expressionusingGram-negativebacteria Shatzman as yet, there are some initial reports that another class of foldases, known as chaperonins, do indeed aid in the production and assembly of multimeric proteins [13 °°]. Chaperonins are a ubiquitous class of proteins that seem to facilitate the folding of other polypeptides and assist in the assembly of oligomeric structures of which they are not a part [14 o]. It has been further proposed that chaperonins prevent the formation of incorrect structures and unscramble those that do occur. Goloubinoff et al. [13 °'] have shown that over-expressing the E. coli GroEL chaperonin proteins at the same time as expressing the A n i d u l a n s ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCo) genes in E. coli led to a five- to tenfold increase in the level of expression of the RuBisCo gene products. In addition, the GroEL products were shown to be necessary for the formation of active dimers and correctly assembled octamers in E. coll. In a later paper, Goloubinoff et aL [15 °] have also shown that chaperonins from a number of sources, including E. coli, yeast, and chloroplasts, can be used in vitro to facilitate the reconstitution of active RuBisCo from unfolded polypeptides. While this is one of the few cases where a protein-mediated refolding has been accomplished, numerous successes have been reported in the last two years in the area of chemically mediated refolding.

Making better use of heterologous genes expressed in E. coli through chemically mediated in vitro refolding It may seem strange at first to include a section on protein refolding in a review on gene expression, however, improved technologies in protein refolding now give us the confidence to express structurally complex proteins in E. coli safe in the knowledge that they are likely to be refolded to a fully active state. Many proteins have now been purified from inclusion bodies and successfully renatured. Inclusion bodies are, in fact, sometimes viewed as being helpful in that they are readily amenable to purification and they may also stabilize proteins that otherwise might be subject to degradation by host proteases [16]. Hoess etal. [17"] have shown that both the human interleukin-2 and retroviral v-myb proteins can be easily isolated from inclusion bodies by treatment of the insoluble products with ion exchange resins. Furthermore, the interleukin-2 recovered by this simple process was shown to be greater than or equal to 30% active, even without attempts to manipulate the redox status of the three cysteine residues. More typically, proteins expressed in E. coliare recovered from insoluble aggregates through the use of denaturants (typically urea or guanidine), detergents, and redox reagents that aid in the formation of appropriate disulfide cross-links. Despite the fact that these treatments can lead to protein modifications and aggregation, numerous successful attempts at refolding complex proteins (proteins which typically have several disulfide crosslinks in

their native structure) expressed in E. colihave now been made. Structurally complicated proteins have often been difficult, if not impossible, to produce in a fully active state when synthesized in E. coll. These proteins do not, in general, fold properly as they are produced and the cysteines do not form the correct disulfide linkages. In the past, attempts to refold these proteins have been unsuccessful or at best unwieldy as extremely dilute solutions were needed to obtain even a low recovery of fully active protein. However, recent advances in protein refolding have made the production of fully active complex proteins in E. coli more of a reality. For example, Halenbeck et al. [18--] have developed a process for the renaturation and purification of recombinant human macrophage colony-stimulating factor (M-CSF; a disulfide-linked dimefic protein containing 18 cysteines), The authors showed that they could purify the protein in a monomeric form after solubilization using urea and subsequent partial purification using size-exclusion chromatography. The protein was then refolded and dimerized using a mixture of oxidized and reduced glutathione. Unlike other proteins where refolding must be done in very dilute solutions (0.1 mg/ml), M-CSF could be successfully refolded at concentrations of up to 0.7 mg/ml. The refolding yield was a remarkable 80%, with a final overall purification yield of fully active 95% pure M-CSF of 25%. Human tissue plasminogen activator, which has 35 cysteines (34 of which are disulfide cross-linked), has also been expressed in an insoluble form in E. coli and successfully renatured to yield a fully active protein, ab belt at a much poorer recovery rate [19"]. Noel and Tsai [20 o] have shown that solubilization of cysteine-rich protein aggregates by S-sulfonation may be a more useful approach than solubilization with urea or guanidine. In their paper they showed that bovine phospholipase A2 (which contains seven disulfide cross-links) could be expressed at high levels in E. coli and renatured in vitro, and that, using this process, 50-100 mg of fully active protein could be obtained per liter of culture. Previously, de Geus et al. [21] had shown a similar approach could be used to renature porcine phospholipase A2 expressed in E. coli with a 30% total yield.

Secretion in E. coli One way to circumvent the problems associated with incorrect folding of heterologous proteins expressed in E. coli might be to secrete the products into the periplasm or medium. The advantages of this system are numerous and include: protection from numerous cytoplasmic proteases, an environment more conducive to proper disulfide formation (i.e. more oxidized); reduced chance of aggregation because there is more space in which to fold in; expression of a protein with the correct amino terminus; reduced cTtotoxicity; and simplified purification if the protein can be released from the pefiplasm by osmotic shock [8..,22 .-24.]. The usefulness of E. coli as a secretion host is still undergoing evaluation. Reports on

7

8

Expressionsystems the successful secretion of foreign proteins in an active form have been relatively rare and a general method to secrete foreign gene products has yet to be established. However, over the last few years our understanding of the mechanisms governing secretion has grown rapidly and led to far more frequent reports of successful secretion of heterologous gene products. While we have known for manyyears that a signal peptide is required for the targeting of a protein to the periplasmic space, we have only recently begun to understand the role that several host proteins and the secreted protein itself play in the transport process. Summers et al. [25 "], using ]3-1actamase chicken muscle triosephosphate isomerase fusions, have shown that as much as the first 14 amino acids after the signal sequence may be involved in defining a region of properties that is critical to the secretability of proteins in E. coil The authors go on to show [26"] that Arg or Lys residues in this region, which have previously been shown to be deleterious to secretion [27], do not necessarily block the export process. Instead, they find that the identity of the amino acids at each position may play a more important role. For example, in the system they were studying, a Lys at position 4 of the signal sequence did not block secretion, while an Arg at that same position did. The authors conclude that it is the ability of the charged residue to become deprotonated or to participate in ion pairs with other amino acid side chains that is the controlling factor in establishing insertion into the inner membrane. At least 25-30 different E. coli genes have now been shown to encode proteins that are involved in the process of protein secretion [28..]. Among the newest members of this class are the chaperonins, which have been discussed earlier in this review with regard to their role in modulating protein folding and assembly. It has now also been shown that three of these proteins, secB, trigger factor and GroEL, can also stabilize the precur sot form of secreted and membrane proteins in conformations that are competent for translocation [29"']. Chaperonins form a far more stable interaction with proteins targeted for secretion than they do with soluble in tracellular proteins and, in doing so, may commit these proteins to the secretory pathway. These stable interactions do not seem to be targeted specifically to signal sequences but rather to hydrophobic regions that may be exposed on the nascent polypeptide chain. Coupled with this improved understanding of secretion mechanisms, some clever molecular biological approaches have led to significant advances in our abilities to secrete heterologous gene products from E. coli. Lunn et al. [30] have developed a now widely used expression system which uses the OmpA leader sequence to direct protein secretion into the periplasmic space of E. coil This system has been used successfully to synthesize and secrete several gene products including subtilisin [ 9 " ] and the normally intracellular superox ide dismutase [24"]. In both cases, the secreted products were shown to be active and the signal sequence precisely removed to leave an authentic N-terminus. Hsuing et al. [31 "'], using a derivative of these OmpA signal

vectors, reported secretion of human growth hormone, not into the periplasm, but into the culture medium. This was accomplished by the co-expression of bacteriocin release protein (BRP), a 28-amino-acid peptide, which upon localization in the host outer membrane activates the detergent-resistant phospholipase thus creating permeable zones in the cell outer membrane through which proteins can pass. By controlling the level of expression of BRP, the rate of permeability can be controlled, resulting in the specific release of periplasmic proteins. In a more mechanistic approach, Stahl and Christiansen [32 "] have selected mutations in signal sequences by screening for clones that had increasing levels of resistance to ampicillin (encoded by the secreted protein lS-lactamase). These improved signal sequences were then used to export proinsulin from E. coliwith a resulting fivefold increase in secretion over that obtained with the wildtype 13-1actamase signal sequence.

Gene expression in other Gram-negative bacteria While most expression efforts in E. coli are aimed at producing heterologous gene products at levels sufficient to facilitate easy purification, altered gene expression in other Gram-negative organisms is more typically aimed at developing new phenotypes through the expression of heterologous genes, or the amplification or deletion of an endogenous gene. This is not to say that high levels of heterologous gene expression are not obtainable in other Gram-negative bacteria. In fact, the Klebsiella aer~> genes urease gene has been expressed in Salmonella p h i m u r i u m at levels approaching 18% of total cellular protein [33"]. The focus on generating altered phenotypes rather than generating high-level expression is a recognition of the potential value of the diverse forms of bacteria that exist in nature, many of which possess attributes that may have industrial applications. In reviewing the genetic engineering of bacteria from managed and natural habitats, Lindow et aL [34"] have outlined several strategies that may be used to modify bacteria for useful purposes. Through the deletion of undesirable traits and/or the transfer of one or more genes from one organism to another, bacteria may be engineered that can better survive their natural or foreign habitats or persist in the presence of toxic agents. For example, there was a well publicized case in which gene expression in P s e u d o m o n a s syringae was altered by the deletion of the gene that encoded the functions responsible for the formation of ice nucleation sites on susceptible plants [35]. When these 'ice minus' bacteria were allowed to colonize plants, the numbers of 'ice plus' colonies that could then form were reduced, thus minimizing frost damage. One of the most active areas of research regarding gene expression in Gram-negative bacteria is the development of bacteria with novel metabolic or catabolic activities produced through the introduction and expression of

Gene expression using Gram-negative bacteria Shatzman 9 heterologous genes. Genetic tools have been used to develop bacteria with specific catabolic pathways that are useful in the degradation of xenobiotics. These bacteria have been further manipulated so that they can function under a wide range of environmental conditions. Many bacterial genes for xenobiotic degradation came from plasmids in strains isolated from contaminated waste sites. For example, a strain of Pseudomonas putida has now been developed (through gene transfer technology) that is capable of degrading phenoxyacetic acid [36"]. Similar gene transfer experiments have improved the economics of using Gram-negative bacteria in commercial processes that produce chemicals and fuels. For example, Zymomonas mobilis has been shown to produce ethanol at a rate three to four times faster and at a higher yield than traditionally used yeast strains. However, this organism is very restricted in the range of substrates it can use as a carbon source (glucose, fructose and sucrose). It has been suggested [37] that Z. mobilis could become the 'ideal ethanol producer' if its narrow substrate range could be overcome. S u e t al. [38 °] have suc cessfully cloned the Xanthomonas albilineans [3-glucosidase gene into Z. mobilis, thus conferring upon it the ability to grow on the cheaper carbon source, cellobiose. The ability to grow on lactose and galactose has also been imparted to Z. mobilis by the transfer of the E. coli lactose and galactose utilization operons [39"]. The review by Lindow etal. [34 ,°] points out that E. colihas also been used extensively for similar gene transfer experiments. Strains of E. coli have been generated that can grow on altered carbon sources [40], degrade toxic chemicals [41] and even remove metals from wastewater [42]. One area where research into altered gene expression in Gram-negative bacteria may rapidly find its way into everyday life is through the development of stable recombinant avirulent Salmonella vaccine strains. Curtiss et al. [43 "'] have recently reviewed progress in the development of avirulent strains that are capable of expressing colonization and virulence antigens from other pathogens. They suggest that such strains could be used to stimulate long-lasting immunity to Salmonella and also to the pathogen supplying the genetic information for these colonization and virulence antigens. For example, Strugnell et al. [44 o] recently reported the expression of the tetanus toxin fragment C following stable integration of the gene into the chromosome of an attenuated strain of Salmonella Ophimurium. Sadoff et al. [45 "'] have taken these types of studies even further by engineering a strain of S. O~phimurium that expresses the circumsporozoite protein of the malaria causing parasite, Plasmodium berghei. Oral immunization of mice with this recombinant, attenuated strain produced cell-mediated immunity and protected the mice from challenge with live sporozoites. It is likely that the next several years will see these approaches expand to the use of other Gram-negative bacteria as vaccine vectors. In summary, while E. coliwill probably remain the Gramnegative organism of choice for high-level expression of heterologous gene products for the next decade, we may expect to see an increasing role for other Gram-negative

bacteria in industrial, environmental and medical applications.

Acknowledgements I thank Christine Debouck, John Keller, Lois Hoyer and Edward Appelbaum for help in editing this manuscript.

Annotated references and recommended reading • •.

Of interest Of outstanding interest

1.

BROSlUS,In Vectors edited by Rodfiguez and Denhardt. Butterworths, 1988, pp205 226.

SEOW HF, GOH CR, KRISHNAH L, PORTER A: Bacterial expression, facile purification and properties of recombinant h u m a n l y m p h o t o x i n ( t u m o r necrosis factor beta). Bit/Technology 1989, 7:363-368. H u m a n TNF-[3 is expressed in a soluble active form in E. coli. Expression levels are optimized by minimizing mRNA secondary structure and encouraging optimal codon utilization. 2. ••

3. •

SCHAUDERB, MCCARTHYJ: The role of bases u p s t r e a m of the Shine-Dalgarno region and in t h e coding s e q u e n c e in the control of gene expression in E. coli: translation and stability of mRNAs in vivo. Gene 1989, 78:59-72. Data are presented indicating that in the presence of a given Shine-Dalgarno start codon combination, the decisive factor for translation initiation efficiency is the stability of base pairing in, or in the vicinity of, this region. 4. ••

DEVLINP, DRUMMOND R, TOY P, MARK D, WATI" K, DEVIJN J: Alteration of amino-terminal c o d o n s of h u m a n granulocytecolony-stimulating factor increases expression levels and allows efficient processing by m e t h i o n i n e aminopeptidase in E. coli. Gene 1988, 65:13-22. Expression of h u m a n G-CSF in E. coli could not be detected when the gene was cloned downstream of either two strong promoters. Decreasing the guanine and cysteine content of the 5' end of the coding region without altering the predicted amino acid sequence increases expression by up to 17% of total soluble protein. 5.

HSIUNGHM, MACKELIARWC: Expression of bovine g r o w t h h o r m o n e derivative in Escherichia coli and t h e use of t h e derivatives to p r o d u c e natural s e q u e n c e g r o w t h horm o n e by cathepsin C cleavage. Methods Enzymol 1987, 153:390-401.

ISACCHIA, SARMIENTOS P, LORENZETTI R, SOmA M: Mature apolipoprotein A1 and its p r e c u r s o r proApoAl: influence of t h e s e q u e n c e at the 5' e n d of t h e g e n e on t h e efficiency of expression in E. coli. Gene 1989, 87:129-137. Changes at the 5' end of the ApoA1 gene could significantly affect the efficiency of expression. However, a causal relationship could not be established between expression levels and codon utilization, base composition, or alterations in secondary structure. 6. ••

7.

SUTIPHONGJ, JOHANSENH, SATHEG, ROSENBERGM, SHATZMANA: Selection of mutations that increase al-antitrypsin g e n e expression in Escherichia coli. Mol Biol Med 1987, 4:307-322.

8. SCHEINC: Production of soluble r e c o m b i n a n t proteins in •• bacteria. Bit~Technology 1989, 7:1141 1149. A review that defines the process of protein folding and suggests approaches that may be used to enhance protein folding in bacteria.

10

Expressionsystems 9. ••

TAKAGIH, MOR1NAGAY, TSUCHIYA M, IKEMURAH, INOUYE M: Control of folding of proteins s e c r e t e d by a high expression secretion vector, pIN-III-Ompka 16-fold increase in production of active subtilisin E in E. coli. 1990, in press. Results are presented that indicate that the rate of protein synthesis and the culture temperature are important factors for obtaining correct folding of proteins being secreted across the cytoplasmic membrane of E. coll. 10. •

SCHEIN C, NOTEBOM M: Formation of soluble r e c o m b i n a n t proteins in E. coli is favoured by lower g r o w t h temperature. Bio/Technology 1988, 6:291-294. Human interferon-c~ is completely insoluble when produced in E. coli at 37°C. However, at growth temperatures of 23~0°C, 30-90% of the recombinant protein was soluble indicating that lower growth temperature favors formation of soluble proteins. 11.

MIZUKAMIT: Production of active h u m a n interferon. Biotech Lett 1987, 8:605-610.

12.

AUGEREA, BENNET GN: T e m p e r a t u r e optimisation of invivo expression from t h e E. coli TRP and TRP:LAC promoters. Biotech Lett 1987, 9:157-162.

ever, this can typically be accomplished only by carefully optimizing every parameter in the refolding process used in renaturation. 20. •

NOELJP, TSAI M-D: Phospholipase A2 engineering: design, synthesis, and expression of a g e n e for bovine (pro) phospholipase A2. J Cell Biochem 1989, 40:309-320. Use of S-Sulfonation may be a more useful approach to sofubilizing cysteine-rich proteins than the use of urea or guanidine. Through this approach, fully active bovine PLA2 was obtained using an E. coli expression system. 21

22. •

OBUKOWICZMG, TURNERMA, WONG EY, TACONw e : Secretion and e x p o r t of IGF-1 in E. coli strain JMIO1. Mol Gen Genet 1988, 215:19-25. Secretion of proteins from E. coli is dependent upon their solubility. Using IGF-1 as an example, it is shown that only soluble proteins can enter the export mechanism. 23.

13. ••

GOLOUBINOFFP, GATENBYAA, LORIMERGH: GroE heat-shock proteins p r o m o t e assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in E. coli. Nature 1989, 337:44-47. A class of proteins, known as chaperonins, is thought to be involved in the post~translational assembly of polypeptides and their assembly into oligomeric structures. GroE is shown to be a m e m b e r of this class of proteins. 14. •

HEMMINGSENSM, ElMS RJ: Molecular chaperones: proteins essential for t h e biogenesis of s o m e macromolecular structures. Trends Biochem Sci 1989, 14:339-342. The authors illustrate that chaperonins are a ubiquitous class of proteins that facilitate the folding of other polypeptides and their subse~ quent assembly into oligomeric structures of which they are not a part. 15. .

GOLOUB1NOFFP, CHRISTELLERJ, GATENBYAA, LORIMERGH: Reconstitution o f active dimeric ribulose bisphosphate carboxylase from an unfolded state d e p e n d s on two chaperonin proteins and Mg-ATP. Nature 1989, 342:8844389. Chaperonins from several sources are shown to be functional in vitro for mediating the assembly of RuBisCo from the unfolded state to properly folded, dimeric molecules. 16.

CHENG Y-S, KWOH DY, KWOH TJ, SOLTVEDT BC, ZIPSER D: Stabilization of a degradable protein by its overexpression in Escherichia coli. Gene 1981, 14:121-130.

17.

HOESS A, ARTHUR A, WANNER G, FANNING E: Recovery of sol-

.

uble, biologically active r e c o m b i n a n t proteins from total bacterial lysates using ion e x c h a n g e resin. Bio/Technology 1988, 6:1214-1217. Treatment of inclusion bodies containing bacterial lysates with ion exchange resins has been found to solubilize three different recombinant proteins. The authors present a method that may be generally applicable to the purification of insoluble recombinant proteins from any expression system. 18. ••

HALENBECKR, KAWASAKIE, WeaN J, KOTHS K: Renaturation and purification of biologically active r e c o m b i n a n t h u m a n m a c r o p h a g e colony-stimulating factor e x p r e s s e d in E. coli. Bio/Technology 1989, 7:710-715. A process has been developed for the renammtion and purification of recombinant h u m a n M-CSF produced in E. coli. The approaches presented in this paper may be helpfifl in designing purification schemes for other cysteine-rich protg'ins produced in E. coli. 19. •

SARMIENTOSP, DUCHESNE M, DENEFLE P, BO1ZIAUJ, FROMAGEN, DELPORTEN, PARKERF, LELLEVREY, MAYAUXJ-F, CARTWRIGHTT: Synthesis and purification of active h u m a n tissue plasminog e n activator from E. coli. Bi~/Technology 1989, 7:495-501. This work demonstrates that even large and complex eukaryotic proteins can be obtained from E. coli in their active conformation. How-

DE GEUS P, VAN DEN BERGH CJ, KUIPERS O, VERHEIJ HM, HOEKSTRA XVPM, DE HAAS GH: Expression of porcine pancreatic phospholipase A2. Generation of active e n z y m e by sequence-specific cleavage of a hybrid protein from Escherichia colg Nucl Acids Res 1987, 15:3743-3759.



LINARDC, MBIKAY M, BENJANNET S, LAZURE C, SEIDAH N, CHRETIENM: Correct processing and secretion of a h u m a n prostatic secretory protein (PSP94) in E. coli. Gene 1988,

73:479-484. Secretion of a eukaryotic protein and correct processing of a eukaryuric leader sequence are demonstrated in E. coli. This paper shows that bacterial sequences are not necessary to enter the bacterial secretion mechanism. 24. •

TAKAHARAM, SAGA[ H, INOUYE S, INOUYE M: Secretion of hum a n superoxlde dismutase in E. coil Bio/Technology 1988, 6:195-198. Results are presented that show that a foreign cytoplasmic protein can be secreted across the inner membrane of E. coli into the periplasmic space yielding an active enzyme. 25. .

SUMMERSR, KNOWLESJ: Illicit secretion of a cytoplasmic protein into t h e periplasm of E. coli requires a signal peptide plau a portion of t h e cognate secreted protein. J Biol Chem 1989, 264:20074--20081. Secretion of triose phosphate isomerase into the periplasm of E. coli requires the signal peptide and at least three residues of the mature ~-lactamase sequence. Up to 12 amino acids of the mature protein are required for optimal secretion, showing that a signal sequence is necessary, but not sufficient, for secretion into the periplasm. 26. •

SUMMERSR, HARRIS C, KNOWLES J: A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in E. coli. J Biol Chem 1989, 264:20082-20088, The finding that an arginine residue in a signal sequence prevents secretion while a lysine does not suggests that basic residues near the mature N-terminus of a secreted protein must be deprotonated if orderly export is to occur. 27.

LI P, BECKWITH J, INOUYE H: Alteration of t h e amino termin u s of t h e m a t u r e s e q u e n c e of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci USA 1988, 85:7685-7689.

28. ••

SAIERM, WERNER P, MULLER M: Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with t h e eukaryotic process. Microbiol Rev 1989, 53:333-366. A review of our current state of knowledge of the molecular mechanisms of protein secretion. The processes and proteins involved are studied and compared in prokaryotes and eukaryotes. 29.

LECKERS, LILLR, ZIEGELHOFFERT, GEORGOPOULOSC, BASSFORD PJ, KUMAMOTOCA, WICKNERW: Three p u r e c h a p e r o n e proteins of E. coli - - SecB, trigger factor and GruEL - - form soluble c o m p l e x e s with p r e c u r s o r proteins. EMBO J 1989, 8:2703-2709. Three chaperonins from E cull have been suggested to interact with precursor proteins in order to maintain them in a conformation which is competent for membrane translocation. The interaction between the • •

Gene expression chaperonins and the secretory precursors is believed to be much more stable than for the interaction with intracellular soluble proteins. 30

LUNN CA, TAKAHARAM, INOUYE M: Use of secretion cloning vectors for guiding the localization of proteins in Escherichia coll. Methods Enzymol 1986, 125:138-149.

31. ••

HSUINGH, CAbrrRELLA. LUIPaNKJ, OUDEGE B, VEROS A, BECKER G: Use of bacteriocin release protein in E. coli for excretion o f h u m a n g r o w t h h o r m o n e into t h e culture medium. Bit~Technology 1989, 7:267-271. Secretion of h u m a n growth hormone into the culture medium rather than the periplasm has been achieved through co-expression of BRP. The authors suggest that this technology could lead to continuous culture of E. coli where cells are immobilized on a solid support and allowed to produce proteins that can be harvested from the media for long periods of time. 32. •

STAHLS, CHRISTIANSENL: Selection for signal mutations that e n h a n c e p r o d u c t i o n of secreted h u m a n proinsulin by E. coli. Gene 1988, 71:147-156. This paper demonstrates the feasibility of using protein fusions as a positive selection for signal sequence mutations that enhance periplasmic levels of foreign proteins. This approach resulted in a fivefold increase in h u m a n pro-insulin expression and secretion in E. coli. 33. •

MULROONEYSB, PANKRATZ S, HAUSINGER RP: Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes urease. J Gen Micro 1989, 135:1769-1776. A wide variety of bacteria can be used to express the urease gene from K aerogenes Of all bacteria studied by these authors, expression in S. O~phimurium was found to be the best when it reached 18% of total soluble protein. 34. . •

LINDOWSE, PANOPOULOSNJ, MCFARLANDBL: Genetic engineering of bacteria from managed and natural habitats. Science 1989, 244:1300-1307. This review focuses on the modification of bacteria that could greatly affect natural and industrial processes. The vast potential for use of bacteria in a large n u m b e r of commercial applications is highlighted. 35.

ORSERC, STASKAWICZBJ, PANOPOULOSNJ, DAHLBECKD, LINDOW SE: Cloning and expression of bacterial ice nucleation g e n e s in Escherichia coli. J Bacteriol 1985, 164:359`366.

36. •

HARKERA, OLSEN R, SELDLERRJ: Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) p a t h w a y of plasmid pJP4: mapping and characterization of t h e TFD regulatory gene, ~dR. J Bacteriol 1989, 171:314-320. A strain of Pseudomonas putida has been engineered that can use a novel carbon energy source. This paper is a fine illustration of the growing ability to engineer Gram-negarive bacteria to metabolize or degrade toxic chemicals. 37.

ESSERK, KARSCHT: Bacterial ethanol production: advantages and disadvantages. Process Biochem 1984, 19:116-123.

using Gram-negative

bacteria Shatzman

38. •

SU P, DE1ANEY S, ROGERS PL: Cloning and expression of a beta glucosidase g e n e from X a n t h o m o n a s a l b i l i n e a n s in E. coli and Z. mobilis. J Biotechnol 1989, 9:139-152. The ability to grow on a new carbon source has been imparted to Z mobilis by inserting the gene cloned from a heterologous bacterium. This could significantly increase the impact of this organism in industrial applications. 39. •

BUCHHOLZ SE, DOOLBY MM, EVELEIGH DE: Growth of Z ~ m o m o n a s on lactose, gene cloning in combination with mutagenesis. J I n d Micro 1989, 4:19-27. The substrate range of Z mobilis has been expanded to include lactose by the introduction of the E. coli lactose operon. 40.

GILBERTHJ, SULLIVANDA, JENKINS G, KEH.FT LE, MINTON NP, HALLJ: Molecular cloning of multiple xylanase g e n e s from P s e u d o m o n a s fluorescens subsp, cellulosa. J Gen Micro 1988, 134:3239`3251.

41.

MONDELLOFJ: Cloning and e x p r e s s i o n in Escherichia coli of P s e u d o m o n a s strain LB400 g e n e s encoding polychlorinated biphenyl degradation. J Bacteriol 1989, 171:1725-1732.

42.

ROMEYERSM: Bit-accumulation of heavy metals in E. coli expressing an indivisable synthetic h u m a n metallothionein gene. J Biotechnol 1988, 8:207-214.

43. , •

CURTIS8 R, KELLY SM, GULIG PA, NAKAYAMAK: Selective delivery of antigens by r e c o m b i n a n t bacteria. Curr Topics Microbiol I m m u n o l 1989, 146:35-49. The way to attenuate Salmonella and to endow such avimlent strains with the ability to express colonization and virulence antigens from other pathogens is reviewed. The potential use of such strains as vaccine delivery systems is also described. 44. •

STRUGNELLR, MASKELLD, FAIRWEATHERN, PICKARDD, COCKAYNE A, PENN C, DOUGAN G: Stable expression of foreign antigens from the c h r o m o s o m e of S a l m o n e l l a t y p h i m u r i u m vaccine strains. Gene 1990, 88:57-63. The stable expression of tetanus toxin C has been achieved in an attenuated strain of S. OJphimurium. The mechanism developed to attenuate this strain, while introducing new genes into the chromosome, should be applicable to the development of a variety of attenuated bacterial strains. 45. ••

SADOFF J, BALLOU WR, BARON LS, MAJARIANXVR, BREY RN, HOCKMEYERWT, LOWELLGH, CHULAYJD: Oral S a l m o n e l l a typ h i m u r i u m vaccine expressing circumsporozoite protein protects against malaria. Science 1988, 240:336-338. A method is described for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated S. Oephimurium transformed with the Plasmodium berghei circumsporozoite gene. This approach suggests the feasibility of developing orally administered vaccines against h u m a n malaria using bacterial vectors.

11

Gene expression using gram-negative bacteria.

Gene expression using Gram-negative bacteria Allan R. Shatzman SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA Current Opinion in B...
874KB Sizes 0 Downloads 0 Views