Sui-Lam Wong

FUNCTIONAL PRODUCTION AND CHARACTERIZATION OF A FIBRIN-SPECIFIC SINGLE-CHAIN ANTIBODY FRAGMENT FROM BACILLUS SUBTILIS: EFFECTS OF MOLECULAR CHAPERONES AND A WALL-BOUND PROTEASE ON ANTIBODY FRAGMENT PRODUCTION

ABSTRACT To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins

During the past year, significant progress has been made using Bacillus subtilis to produce a wide range of foreign proteins. Through strain improvement and co-expression of molecular chaperones, secretory proteins can be produced at a higher level. Through protein engineering, target proteins can be redesigned to have better stability and solubility. A combination of these two strategies would be a useful approach to produce heterologous proteins from B. subtilis at high quality and with a high yield.

High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis

Staphylokinase is a promising blood-clot dissolving agent for the treatment of patients suffering from a heart attack. It would be desirable to produce this protein in large quantities for biochemical characterization and clinical trials. Production of intact, biologically active staphylokinase from bacterial expression systems has been a challenge because of N-terminal microheterogeneity, plasmid instability, or low-production yield. By using a seven-extracellular-protease deficient Bacillus subtilis strain, WB700, intact staphylokinase can be produced via secretion. However, native staphylokinase gene (sak) in a high-copy number plasmid was found to be unstable in B. subtilis. To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of sak were replaced by B. subtilis promoters (P43, a constitutively expressed promoter; Pamy, a stationary-phase promoter; and PsacB, a sucrose-inducible promoter) and the levansucrase-signal sequence, respectively. This overcame the plasmid instability problem. To enhance transcription from the sacB promoter, degQ encoding a transcriptional activator for sacB and other protease genes was also installed in the expression vector. The use of WB700 as the expression host allowed enhanced production of staphylokinase from the sucrose-inducible plasmid without causing any obvious degradation of staphylokinase. Both the P43 and PsacB (with DegQ) promoters worked well. Over 90% of staphylokinase synthesized can be secreted effectively. With the optimization of both the culture media and the fermentation conditions, production of staphylokinase reached a level of 337 mg/L, and staphylokinase could be purified to homogeneity by a simple three-step purification scheme. Secreted staphylokinase did not show any N-terminal heterogeneity. This presents an attractive system for the production of staphylokinase in both high quality and quantity.

Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a β-lactamase fusion protein

A secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.

Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800.

ABSTRACT Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.

Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins.

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.

The role of the pro‐sequence in the processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus

The Bacillus cereus cnp gene coding for the thermolysin‐like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre‐pro‐protein (Mr= 61000); the pro‐peptide is approximately two‐thirds of the size of the mature protein. The pro‐sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro‐sequences are shown to be homologous to the pro‐sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro‐protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro‐sequences. The detection results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N‐terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro‐sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro‐protein.

Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation

The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon “intercellular complementation.” Interesting implications concerning bacterial cooperation are suggested from these results.

Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis

pUb110-based vectors are commonly used for expression and secretion studies in Bacillus subtilis. Two of these plasmids, pUB18P43 and pWB705, have been applies to produce several proteins of interest. To offer greater flexibility and compatibility in this system, the authors have also constructed a pE194-based plasmid vector (pE18). To determine whether the pUB110-based or the pE194-based vector serves as a better expression system, three structural genes encoding cytoplasmic BirA, extracytoplasmic PrsA and extracellular staphylokinase, respectively, were used as models. Production of these products using pUB110-based vectors was consistently 2--3-fold lower than that using the pE194-based vectors. The observed difference in the protein yield did not result from either the rearrangement of the plasmid or the difference in the plasmid copy number. By using three different approaches, the lower production yield from the pUB110-based vectors was found to be due to the transcription interference from the plasmid encoded genes. These findings illustrate that the orientation of the inserted gene in pUB110-based vector can greatly affect gene expression. Two new expression vectors, pUB19P43 and pWB980, were constructed to allow better gene expression.

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

ABSTRACT Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.